Retroviruses
Retroviruses are RNA viruses. When a host cell is infected with a retrovirus, the genomic RNA is reverse transcribed into a DNA intermediate which is integrated very efficiently into the chromosomal DNA of infected cells. The integrated DNA intermediate is referred to as a provirus. The family Retroviridae are enveloped single-stranded RNA viruses that typically infect mammals, such as, for example, bovines, monkeys, sheep, and humans, as well as avian species.
Retro Viral Envelope Proteins
Retroviruses carry their genomes as two copies of a single RNA molecule and the simplest retroviruses contain the gag, pro, pol and env genes.
The first step in the replication cycle of a retrovirus is its entry into a host cell (see FIG. 1). The envelope protein (env) is responsible for binding of the retrovirus to a specific cell surface receptor. A retroviral receptor is a membrane integral protein in the plasma membrane of the host cell and as such has a function unrelated to virus infection.
However, retroviral envelopes that use non-protein receptors are known, e.g., the vesicular stomatitis virus.
Retroviruses can be thought of as a protein-package comprising RNA wrapped in a lipid membrane that contains glycoproteins. The lipid bi-layer is derived from the cell membrane after budding and is thought to be associated with a viral gene product, a peripheral membrane protein called Matrix (MA). Traversing through the lipid bi-layer is another viral gene product, the envelope protein, which consists of two subunits: the transmembrane (TM) and the surface unit (SU). The function of the envelope protein is binding of the virus to its target cell and mediating fusion of the viral and cellular membranes.
The retroviral envelope protein can be seen as a nano-device that mediates receptor-dependent fusion of biological membranes. When the envelope protein is attached to the lipid-bilayer membrane surrounding the virus, the net result of fusion with a cellular membrane is entry of the nucleoprotein core of the virus into the cytoplasm. Such fusion is triggered by the envelope protein's recognition of a receptor on the plasma membrane or an endosomal membrane. Natural receptors for retroviral infection are integral membrane proteins with multiple membrane-spanning domains. For the gammaretroviruses such as murine leukemia viruses, several natural receptors are known to have transporter functions for e.g. amino acids. When expressed on the plasma membrane of a cell, the viral envelope protein may also mediate cell to cell fusion. The dynamics of the fusion process is generated by the viral envelope protein which is produced in an activated state and has “one shot” to trigger membrane fusion.
The ability of redirecting the retroviral fusion machinery to a desired receptor would have wide biotechnological and potentially also nanotechnological applications. However, the regulatory mechanisms that interconnect receptor binding with fusion are poorly understood, which has made intelligent engineering of the envelope protein difficult. Many attempts at redirecting the receptor-specificity have found that incorporation of a ligand into the envelope protein may cause receptor-dependent binding without activation of the fusion machinery.
SL3-2 Murine Leukaemia Virus Envelope Polypeptide
In an amino acid sequence alignment between SL3-2 and MCF-247, a region has been found to display differences in the 15 amino acids long stretch upstream of the proline rich region. This region has been named VR3 by the present inventors. Further, a sequence alignment of MLVs from different sub-families show conserved amino acids at positions 203-208 WGLRLY and at positions 214-215 DP based on SL3-2 sequence, thus defining a 13 amino acid stretch (see FIGS. 4-5).
In the present context, the term “VR3 region” comprises all of the amino acids found between the residue found at two positions after the conserved tryptophan 197 and the residue before the conserved aspartic acid 214 (according to the sequence shown in SEQ ID NO:2) including these two positions.
Tropism of Murine Leukaemia Virus (MLV)
The MLVs are a group of gammaretroviruses that has been divided into families based on their host range and interference properties. The families are the ecotropic, amphotropic, xenotropic and polytropic subfamilies. Ecotropic viruses are defined by their usage of the mCAT-1 receptor (Wang et al. 1991). Ecotropic viruses are able to infect only murine cells. Examples of ecotrpic viruses are Moloney MLV and AKV. Amphotropic viruses infect murine, human and other species through the Pit-2 receptor (Kavanaugh et al. 1994). One example of an amphotopic virus is the 4070A virus. Xenotropic and polytropic viruses utilize the same (Xpr1) receptor. However, the xenotropic and polytropic viruses differ in their species tropism. Xenotropic viruses such as NZB-9-1 infect human and other species but not murine species, whereas polytropic viruses infect murine, human and other species as exemplified by the mink cell focus-forming viruses (MCF) for example the MCF 247 virus. However, the polytropic SL3-2 virus has a host range as the mouse ecotropic viruses in that it infects and replicates in mouse cells, but are impaired in its ability to infect and replicate in mink cells or human cells. The SL3-2 envelope protein virus utilizes the polytropic (Xpr1) receptor.
Retroviral Vectors in Therapy
Retroviral vector particles are useful agents for introducing polynucleotides into cells, such as eukaryotic cells. The term “introducing” as used herein encompasses a variety of methods of transferring polynucleotides into a cell, such methods including transformation, transduction, transfection, and transinfection.
Retroviruses typically have three common open reading frames, gag, pol, and env, which encode the structural proteins, encode enzymes including reverse transcriptase, and encode envelope proteins, respectively. Typically, retroviral vector particles are produced by packaging cell lines that provide the necessary gag, pol, and env gene products in trans. (Miller, et al., Human Gene Therapy, Vol. 1, pgs. 5-14 (1990)). This approach results in the production of retroviral vector particles which transduce mammalian cells, but are incapable of further replication after they have integrated into the genome of the cell.
Thus, retroviral vector particles have been used for introducing polynucleotides into cells for gene therapy purposes. In one approach, cells are obtained from a patient, and retroviral vector particles are used to introduce a desired polynucleotide into the cells, and such modified cells are returned to the patient with the engineered cells for a therapeutic purpose. In another approach, retroviral vector particles may be administered to the patient in viva, whereby the retroviral vector particles transduce cells of the patient in vivo. Chimeric retroviruses have also been suggested in order to induce immune reactions against viruses, however no positive data have been reported showing this effect in humans.
Viral Interference
Among viruses such as the murine γ-retroviruses a phenomenon termed receptor interference has been used to classify viruses based on their tropism (Sommerfelt et al. 1990). Upon infection the virus synthesize de novo envelope proteins for the production of new viral particles. Some of these envelope proteins will engage the receptor via an unknown mechanism and shield the receptor (FIG. 2). This shielding prevents the recurrence of an infective event by an exogenous virus. In cell culture the interference is very effective in that complete block of infection can be observed.
HIV-1 is somewhat different with regard to receptor usage. For HIV-1 entry to occur a two-step binding mechanism is required. First the HIV-1 envelope protein binds the CD4 receptor (primary receptor) (Eckert et al 2001). This event initiates a conformational change that exposes a region termed V3 (Variable loop 3) which is responsible for a second interaction with a co-receptor (either CCR-5 or CXCR-4) (Huang et al 2005). This co-receptor interaction is absolutely required for infection to occur. In cell culture the same degree of receptor interference is not observed by HIV-1 infection, which may be due to the dual receptor requirement.
The retroviral phenomenon of superinfection resistance (SIR) defines an interference mechanism that is established after primary infection, preventing the infected cell from being superinfected by a similar type of virus.
In most cases, virus-encoded proteins are responsible for the phenomenon of SIR. A simple form of SIR is receptor occupancy by viral Env proteins, preventing the binding of a second virus, but many additional mechanisms have been described. SIR is furthermore not restricted to retroviruses.
Uses of Chimeric Retroviral Envelopes
Ecotropic and amphotropic MLVs have been widely used as research tools. Ecotropic viruses are usually chosen because of safety concerns, while the amphotropic viruses have the ability to infect human cells. Different packaging cell lines that express the ecotropic or amphotropic envelopes have been designed to fulfil these different requirements.
Several functional chimeric envelopes have already been described but none of these can mediate transduction at efficiencies comparable to the efficiencies obtained with wild type envelope proteins. The described functional chimeric MLV-envelopes can be divided into two groups. The first group has the heterologous ligand inserted in the N-terminal of the SU-protein and can mediate transduction without co-expression of wild type envelope, whereas the other group has the ligand inserted internally in SU and is dependent of co-expressed wild type envelope. Peptide linkers and a single chain antibody specific for the human major histocompatibility complex class I(MHC-I) molecule have e.g. been inserted at four internal positions in Akv-env.
The first attempts to direct virus particles towards receptors not normally recognised by retroviruses were done by antibody-bridging and by usage of chemical modifications. By cross-linking monoclonal antibodies against SU and the transferring receptor with a sheep anti-mouse kappaiight chain antibody binding of the virus to human HEp2 cells, and subsequent internalisation was shown. However, internalisation of the virus by this infection route was not followed by establishment of the proviral state.
Others used a similar approach to target the attachment of ecotropic viruses by streptavidin bridging biotinylated antibodies against SU and against specific membrane markers expressed on human cells. By this method human cells expressing MHC class I, MHC class II, epidermal growth factor and insulin were successfully infected, whereas this method did not prove feasible for promoting infection of cells expressing transferrin, high density lipoprotein and galactose receptors.
Also, chemically coupled galactose residues to ecotropic Env, making the virus particles capable of infecting human hepatoma cells through the asialoglycoprotein receptor, have been tried.
Infection of human cells by an ecotropic virus displaying chimeric-envelope proteins on the surface of the virion is also known to a person skilled in the art. This can be achieved by e.g. substituting a part of MoMLV SU with a sequence encoding the erythropoietin hormone (EPO), insertion of a sequence encoding human heregulin for infection of human breast cancer cells overexpressing the human epidermal growth factor receptor, substitution of an internal fragment of SU with a single-chain variable fragment (ScFv) derived from a monoclonal antibody recognising the human low density lipoprotein receptor which gave a chimeric envelope capable of infecting human cells.
In these reports with chimeric envelopes, targeted infection was only obtained when wild type env was co-expressed with the chimeric construct from the packaging cell line.
This indicates that functional domains are contained within the ecotropic envelope, which is necessary for mediating infection beyond the point of receptor binding.
The obtained targeting efficiencies with chimeric envelopes reported until now are considerably lower than the efficiencies obtained with wild type envelopes. The reasons for these low transduction efficiencies of target cells are probably diverse, including the choice of insertion site, stability of the chimeric envelope protein, the tertiary protein structure and the choice of target cells. Furthermore, the choice of ligand is probably also very important for obtaining infection, as several chimeric envelopes have failed to promote infection. One more positive example relates to insertion of a short nondisruptive peptide (RDG) known to bind to several integrins displayed on the surface of cells (Golan T J and Green-M R, 2002).
The above-described examples all utilised the ecotropic envelope. One advantage of using this envelope is that it is restricted in infecting human cells as the surface protein part of the envelope does not recognise a human receptor. The concept is that if the envelope can be engineered to bind to a human receptor by inserting a heterologous sequence in the envelope mediating this binding, the otherwise intact fusogenic properties of the envelope would mediate the fusion.
Retroviruses
Retroviruses are RNA viruses wherein the viral genome is RNA. When a host cell is infected with a retrovirus, the genomic RNA is reverse transcribed into a DNA intermediate which is integrated very efficiently into the chromosomal DNA of infected cells. The integrated DNA intermediate is referred to as a provirus. The family Retroviridae are enveloped single-stranded RNA viruses that typically infect mammals, such as, for example, bovines, monkeys, sheep, and humans, as well as avian and murine species. Retroviruses are unique among RNA viruses in that their multiplication involves the synthesis of a DNA copy of the RNA which is then integrated into the genome of the infected cell.
The Retroviridae family comprises a number of retroviruses such as the lentiviruses exemplified by HIV-1, HIV-2 and SIV, and the gammaretroviruses such as the leukaemia viruses for example murine leukaemia viruses (MLVs), and feline leukaemia viruses.
Retroviruses are defined by the way in which they replicate their genetic material. During replication the RNA is converted into DNA. Following infection of the cell a double-stranded molecule of DNA is generated from the two molecules of RNA which are carried in the viral particle by the molecular process known as reverse transcription. The DNA form becomes covalently integrated in the host cell genome as a provirus, from which viral RNAs are expressed with the aid of cellular and/or viral factors. The expressed viral RNAs are packaged into particles and released as infectious virion.
The retrovirus particle is composed of two identical RNA molecules. Each wild-type genome has a positive sense, single-stranded RNA molecule, which is capped at the 5′ end and polyadenylated at the 3′ tail. The diploid virus particle contains the two RNA strands complexed with gag proteins, viral enzymes (pol gene products) and host tRNA molecules within a ‘core’ structure of gag proteins. Surrounding and protecting this capsid is a lipid bilayer, derived from host cell membranes and containing viral envelope (env) proteins. The env proteins bind to a cellular receptor for the virus and the particle typically enters the host cell via receptor-mediated endocytosis and/or membrane fusion.
After the outer envelope is shed, the viral RNA is copied into DNA by reverse transcription. This is catalyzed by the reverse transcriptase enzyme encoded by the pol region and uses the host cell tRNA packaged into the virion as a primer for DNA synthesis. In this way the RNA genome is converted into the more complex DNA genome.
The double-stranded linear DNA produced by reverse transcription may, or may not, have to be circularized in the nucleus. The provirus now has two identical repeats at either end, known as the long terminal repeats (LTR). The termini of the two LTR sequences produces the site recognized by a pol product—the integrase protein—which catalyzes integration, such that the provirus is always joined to host DNA two base pairs (bp) from the ends of the LTRs. A duplication of cellular sequences is seen at the ends of both LTRs, reminiscent of the integration pattern of transposable genetic elements. Integration is thought to occur essentially at random within the target cell genome. However, by modifying the long-terminal repeats it is possible to control the integration of a retroviral genome.
Transcription, RNA splicing and translation of the integrated viral DNA is mediated by host cell proteins. Variously spliced transcripts are generated. In the case of the human retroviruses HIV-1/2 and HTLV-I/II viral proteins are also used to regulate gene expression. The interplay between cellular and viral factors is important in the control of virus latency and the temporal sequence in which viral genes are expressed.
Murine Leukaemia viruses are a family of simple retroviruses isolated from laboratory mice. Retroviruses carry their genomes as two copies of a single RNA molecule and the simplest retroviruses contain the gag, pro, pol and env genes. These genes are found in the same order in all known retroviruses, reflecting the phylogenetic relationship of retroviruses.
Retroviral integration can activate genes in the vicinity of the integration site. In this way, retroviruses have been used to identify oncogenes since activation of these genes result in tumour growth. In much the same way the integration of a provirus can disrupt the expression of genes, hence inactivation of a tumour suppressor gene may contribute to tumour formation. A high number of integrations are desirable in such studies since not all integrations result in tumour generation and multiple hits are required. Very few integration events are expected to be near oncogene or tumour suppressor genes. Tumour formation might also involve multiple gene regulations.
Retroviral infections usually result in a single integration event since the envelope protein blocks receptors on an infected cell. This is the basis of the superinfection resistance (also called interference) phenomenon in which a virus-infected cell shows resistance to superinfection by viruses, which utilise the same receptor for entry. Thus, use of viruses with different receptor usage increases the number of integration events. Entry by different receptors may even provide access to retroviral disease induction in different mouse tissues.
The integration mechanism of retroviruses can be used to introduce any DNA sequence into a host genome, if the appropriate cis elements of the retroviral genome are maintained in the transducing vector and the DNA sequence can be encompassed in the vector (less than 9000 bp). Therefore retroviral vectors are attractive tools for gene therapy. Most simple retroviral receptors are found on many different cell types of the same species. That is why vector systems utilising wild type envelopes from simple retroviruses cannot be used to introduce genes in a selective manner into specific cells/tissues.
The retroviral envelope protein is a nano-device that mediates receptor-dependent fusion of biological membranes. When the envelope protein is attached to the lipid-bilayer membrane surrounding the virus, the net result of fusion with a cellular membrane is entry of the nucleoprotein core of the virus into the cytoplasm. Such fusion is triggered by the envelope protein's recognition of a receptor on the plasma membrane or an endosomal membrane. Natural receptors for retroviral infection are integral membrane proteins with multiple membrane-spanning domains. For the gammaretroviruses such as murine leukemia viruses, several natural receptors are known to have transporter functions for e.g. amino acids. When expressed on the plasma membrane of a cell, the viral envelope protein may also mediate cell to cell fusion. The dynamics of the fusion process is generated by the viral envelope protein which is produced in an activated state and has “one shot” to trigger membrane fusion.
The ability of redirecting the retroviral fusion machinery to a desired receptor would have wide biotechnological and potentially also nanotechnological applications. However, the regulatory mechanisms that interconnect receptor binding with fusion are poorly understood, which has made intelligent engineering of the envelope protein difficult. Many attempts at redirecting the receptor-specificity have found that incorporation of a ligand into the envelope protein may cause receptor-dependent binding without activation of the fusion machinery.
Several functional chimeric envelopes have already been described but none of these can mediate transduction at efficiencies comparable to the efficiencies obtained with wild type envelope proteins. The described functional chimeric MLV-envelopes can be divided into two groups. The first group has the heterologous ligand inserted in the N-terminal of the SU-protein and can mediate transduction without co-expression of wild type envelope, whereas the other group has the ligand inserted internally in SU and is dependent of co-expressed wild type envelope. Peptide linkers and a single chain antibody specific for the human major histocompatibility complex class I(MHC-I) molecule have e.g. been inserted at four internal positions in Akv-env.
The first attempts to direct virus particles towards receptors not normally recognised by retroviruses were done by antibody-bridging and by usage of chemical modifications. By cross-linking monoclonal antibodies against SU and the transferring receptor with a sheep anti-mouse kappa light chain antibody binding of the virus to human HEp2 cells, and subsequent internalisation was shown. However, internalisation of the virus by this infection route was not followed by establishment of the proviral state.
Others used a similar approach to target the attachment of ecotropic viruses by streptavidin bridging biotinylated antibodies against SU and against specific membrane markers expressed on human cells. By this method human cells expressing MHC class I, MHC class II, epidermal growth factor and insulin were successfully infected, whereas this method did not prove feasible for promoting infection of cells expressing transferrin, high density lipoprotein and galactose receptors.
Also, chemically coupled galactose residues to ecotropic Env, making the virus particles capable of infecting human hepatoma cells through the asialoglycoprotein receptor, have been tried.
Infection of human cells by an ecotropic virus displaying chimeric-envelope proteins on the surface of the virion is also known to a person skilled in the art. This can be achieved by e.g. substituting a part of MoMLV SU with a sequence encoding theerythropoietin hormone (EPO), insertion of a sequence encoding human heregulin for infection of human breast cancer cells overexpressing the human epidermal growth factor receptor, substitution of an internal fragment of SU with a single-chain variable fragment (ScFv) derived from a monoclonal antibody recognising the human low density lipoprotein receptor which gave a chimeric envelope capable of infecting human cells.
In these reports with chimeric envelopes, targeted infection was only obtained when wild type env was co-expressed with the chimeric construct (from thet1) 2 packaging cell line). This indicates that functional domains are contained within the ecotropic envelope, which is necessary for mediating infection beyond the point of receptor binding.
The obtained targeting efficiencies with chimeric envelopes reported until now are considerably lower than the efficiencies obtained with wild type envelopes. The reasons for these low transduction efficiencies of target cells are probably diverse, including the choice of insertion site, stability of the chimeric envelope protein, the tertiary protein structure and the choice of target cells. Furthermore, the choice of ligand is probably also very important for obtaining infection, as several chimeric envelopes have failed to promote infection. One more positive example relates to insertion of a short nondisruptive peptide (RDG) known to bind to several integrins displayed on the surface of cells (Golan T J and Green-M R, 2002).
The above-described examples all utilised the ecotropic envelope. One advantage of using this envelope is that it is restricted in infecting human cells as the surface protein part of the envelope does not recognise a human receptor. The concept is that if the envelope can be engineered to bind to a human receptor by inserting a heterologous sequence in the envelope mediating this binding, the otherwise intact fusogenic properties of the envelope would mediate the fusion.
The present invention provides improved chimeric envelope proteins with novel ligands and ligand insertion sites within the envelope polypeptide that are advantageous over prior art chimeric envelopes, for example in relation to improving therapeutic efficacy of gene therapies.